Site-specific experiments on folding/unfolding of Jun coiled coils: Thermodynamic and kinetic parameters from spin inversion transfer nuclear magnetic resonance at leucine-18

D. André D'Avignon, G. Larry Bretthorst, Marilyn Emerson Holtzer, Kathleen A. Schwarz, Ruth Hogue Angeletti, Lisa Mints, Alfred Holtzer

Research output: Contribution to journalArticle

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Abstract

The 32-residue leucine zipper subsequence, called here Jun-lz, associates in benign media to form a parallel two-stranded coiled coil. Studies are reported of its thermal unfolding/folding transition by circular dichroism (CD) on samples of natural isotopic abundance and by both equilibrium and spin inversion transfer (SIT) nuclear magnetic resonance (NMR) on samples labeled at the leucine-18 α-carbon with 99% 13C. The data cover a wide range of temperature and concentration, and show that Jun-lz unfolds below room temperature, being far less stable than some other leucine zippers such as GCN4. 13C-NMR shows two well-separated resonances. We ascribe the upfield one to 13C spins on unfolded single chains and the downfield one to 13C spins on coiled-coil aimers. Their relative intensities provide a measure of the unfolding equilibrium constant. In SIT NMR, the recovery of the equilibrium magnetization after one resonance is inverted is modulated in part by the unfolding and folding rate constants, which are accessible from the data. Global Bayesian analysis of the equilibrium and SIT NMR data provide values for the standard enthalpy, entropy, and heat capacity of unfolding, and show the latter to be unusually large. The CD results are compatible with the NMR findings. Global Bayesian analysis of the SIT NMR data yields the corresponding activation parameters for unfolding and folding. The results show that both reaction directions are activated processes. Activation for unfolding is entropy driven, enthalpy opposed. Activation for folding is strongly enthalpy opposed and somewhat entropy opposed, falsifying the idea that the barrier for folding is solely due to a purely entropic search for properly registered partners. The activation heat capacity is much larger for folding, so almost the entire overall change is due to the folding direction. This latter finding, if it applies to GCN4 leucine zippers, clears up an extant apparent disagreement between folding rate constants for GCN4 as determined by chevron analysis and NMR in differing temperature regimes.

Original languageEnglish (US)
Pages (from-to)255-267
Number of pages13
JournalBiopolymers
Volume83
Issue number3
DOIs
Publication statusPublished - Oct 15 2006

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Keywords

  • Coiled coils
  • Leucine zipper
  • Protein denaturation
  • Protein folding
  • Spin inversion transfer

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Biomaterials
  • Organic Chemistry

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